Insulin independence among patients with diabetes utilizing an optimized hamster reg3 gamma peptide

ABSTRACT

Embodiments of the present invention provide for novel therapies, pharmaceutical compositions and methods for insulin independence utilizing a new optimized hamster Reg3 gamma peptide, which is new to the art and has not previously been considered for development in the 30 year history since its discovery. Methods, pharmaceutical compositions and therapies novel to the prior art are utilized in this invention to render patients with recent onset and existing type 1 diabetes insulin independent by an optimized hamster Reg3 gamma peptide and an immune tolerance agent for type 1 patients to become insulin independent and used alone without an immune tolerance agent for type 2 diabetes. While not wishing to be bound by theory, optimized Reg3 gamma peptides increases beta cell generation by its demonstrated properties shown within of transforming ductal pancreatic cells into new islets.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.14/453,414, now U.S. Pat. No. 9,321,812, filed on Aug. 6, 2014,entitled, “INSULIN INDEPENDENCE AMONG PATIENTS WITH DIABETES UTILIZINGAN OPTIMIZED HAMSTER REG3 GAMMA PEPTIDE,” which relies on the disclosureof and claims the benefit of priority to the filing date of U.S. Ser.No. 61/971,721, filed on Mar. 28, 2014. This application is also acontinuation application of U.S. Ser. No. 14/453,421, filed on Aug. 6,2014, which relies on the disclosure of and claims the benefit ofpriority to the filing date of U.S. Ser. No. 61/971,721, filed on Mar.28, 2014. Each of the foregoing patent applications, patentpublications, and patents is hereby incorporated by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED IN COMPUTER READABLE FORM

The present application contains a Sequence Listing which has beensubmitted in ASCII format by way of EFS-Web and is hereby incorporatedby reference herein in its entirety. The ASCII file was created Jul. 17,2014 and named CLEV012seq.txt, which is 2.90 kilobytes in size and whichis identical to the paper copy filed with this application.

FIELD OF THE INVENTION

The present invention relates to novel therapies, pharmaceuticalcompositions and methods for treating conditions that are associatedwith or are a risk factor for impaired glucose homeostasis includingtype 1 and 2 diabetes utilizing an optimized hamster Reg3gamma 15-aminoacid peptide.

BACKGROUND OF THE INVENTION

Diabetes is one of the most serious health issues facing humanity withThe World Health Organization reporting that approximately 346 millionpeople worldwide have already been diagnosed with diabetes, making it aglobal challenge. Diabetes is a chronic disease that manifests wheninsulin production by the beta cells of the pancreas is insufficient.Type 1 and type 2 diabetes have long been considered diseases resultingfrom diminished insulin secretion. Research carried out over the pastcentury has more clearly found that generating new beta cells that makeinsulin is the key to reversing this disease.

Beta cells, which secrete insulin, were discovered in 1869 by a medicalstudent, Paul Langerhans. Pancreatic islets, which are predominatelycomprised of beta cells, are highly active metabolically, utilizing 20%of the blood supply delivered to the pancreas, but only accounting for2% of the pancreatic mass; the remainder being extra-islet exocrinetissue containing ductal, acinar and progenitor tissue.

Among type 2 diabetes patients, there is a 50-80% reduction in beta cellmass by the time of diagnosis compared to a reduction in beta mass by90% or more among type 1 patients, who commonly have an autoimmunecomponent to their beta cell loss. Although the beta cell mass mayexpand several fold from birth to adulthood, this is not enough tocompensate for the greater loss than generation of new beta cells seenin both type 1 and 2 diabetes.

Two recent NIH studies, one in children and adolescents and the other inadults demonstrate that intensive lifestyle interventions designed toimprove and impact type 2 diabetes simply have no effect in children andadolescents and cannot be sustained over time among adults. The TODAYStudy Group. N Engl J Med. 2012 Apr. 29. [Epub ahead of print]. DiabetesResearch Program Prevention Group, Lancet. 2009; 374(9702): 1677-1686.Among children and adolescents with type 2 diabetes, therapy withmetformin or lifestyle interventions did not improve diabetes control orthe necessity for insulin therapy.

The TODAY study illustrates the need for new insulin-secreting betacells to delay or prevent the adverse vascular complications ofdiabetes. Despite the many new treatment and technologicalarmamentariums for diabetes, diabetes-related complications includingretinopathy, blindness, neuropathy, amputations, renal insufficiency anddialysis, along with macrovascular complications including heart attack,stroke and peripheral vascular disease have risen among patients withdiabetes. For example, recent studies among patients with type 1utilizing advances including the use of glucose sensors and insulinpumps did not improve hemoglobin A1C levels as much as those seen in theDCCT trial conducted more two decades ago when there were much morelimited treatment options. The DCCT Research Group. N Engl J Med. 1993;329(14):977-986, Bergenstal R M et al, N Engl J Med. 2010;363(4):311-320. Bergenstal R M, et al, Diabetes Care. 2011;34(11):2403-2405.

There is a dire need to restore new beta cells and maintain beta cellmass among type 1 and type 2 diabetes. The loss of endogenous insulin isdirectly correlated with a multiplicity of atherogenic risk factors formicrovascular and macrovascular complications. Lack of insulin, which isthe hallmark of diabetes results not only in elevated glucose levels,but also results in a large number and wide complexity of metabolicabnormalities. For example, lack of insulin results in diminishedactivation of lipoprotein lipase resulting in increased levels oftriglyceride-rich lipoproteins including chylomicrons and verylow-density lipoproteins.

Among type 1 patients the pathology is more complicated, because despiteknown autoimmune attack on the beta cells, the delivery of agents toprotect the beta cells from further attack has not rendered patientswith sustained freedom from exogenous insulin. Despite dozens ofclinical trials with a large variety and types of autoimmune therapiesthat were successful in reversing diabetes in non-obese diabetic (NOD)mice, autoimmune therapy alone provided to patients with type 1 diabeteswithin 3 months of their diagnosis did not sustain insulin-independencesince in man, as compared to mice, there is not the significant betacell regeneration to sustain insulin independence. Some trials withimmune tolerance agents within the first months of diagnosis haverendered 67.5% of patients insulin-free within 7 weeks of therapy, yetover time, all require insulin again.

The leading hypothesis of how new beta cells can be formed in bothchildren and adults is based upon the original works of scientistsnearly a century ago who identified that in acute pancreatic injurythere is new beta cell growth. Frederick Banting discovered insulin in1921 by clamping the pancreatic ducts to induce the formation of newpancreatic cells. Dr. Banting collected the pancreatic secretions afteracute pancreatic ligation and these secretions became known as insulin.Banting F G and Best C H. J Lab Clin Med. 1922; 7:464-472. This work wassupported by several earlier scientists who described that although thepopulation of beta cells is primarily formed during embryogenesis, thereis the ability to grow new beta cells post-natally through a process oftransformation of ductal cell tissue into insulin-producing tissue. By1920, the regenerative powers of the pancreas were well described.Frederick Banting attributes his studies leading to the discovery ofinsulin on the work of Moses Barron who documented that regeneration ofinjured pancreatic tissue manifests from the pancreatic ducts. Barron M.Surg Gynec Obstet. 1920; 19:437-448. Prior to the widespreadavailability of insulin, surgeons performed partial pancreatectomies ondiabetic children in the hopes of stimulating beta cell regeneration.DeTakats G. Endocrinology. 1930; 14:255-264. Benefits from these novelprocedures were described, but were short-lived, likely because ofongoing autoimmune destruction.

Utilizing the data available from the Human Genome Project, thisinventor and others have shown the ability to generate fully-functionalpancreatic beta cells through the differentiation of non-endocrinecells. The ability of bioactive regions of the Reg gene proteins totransform extra-islet ductal tissue into islets has now been shown bymore than a dozen research groups including The Section of Islet Celland Regenerative Biology at Joslin Diabetes Center at Harvard Universityand The Departments of Beta Cell Regeneration at the Hagedorn ResearchInstitute in Denmark. The Reg gene peptides identified by this inventorand others are still in development.

This inventor has previously shown that the human Reg gene peptides aredirectly involved in new beta cell formation from extra-islet ductaltissue. Others have confirmed the presence of Reg in the pancreas ofnewly diagnosed human diabetes, with subsequent data in both humanductal tissues and from BrdU studies showing that Reg serves to directlyform new beta cells from extra-islet ductal tissue. Levetan C S et al,Endocr Pract. 2008; 14(9):1075-1083, Rosenberg L et al, Diabetologia.1996; 39:256-262, Li J et al, Peptides. 2009; 30(12):2242-2249.

The 15-amino acid hamster Reg3 gamma peptide, first identified in 1983and identified as being a hamster Reg3gamma peptide in 2007 (BriocheBiopsy's Act 2007, 1769(9-10):579-85), has not been tolerated in man dueto the high quantity needed and the production of local side-effectsresulting in ⅓ or more of study subjects dropping out of the clinicaltrials (Dungun K M et al, Diabetes Metal Res Rev. 2009; 25(6):558-565).Previously, this inventor demonstrated that a human Reg3a gene proteinhas successfully been administered to human pancreatic ductal tissuedevoid of islets resulting in a significant increase in insulinconcentrations indicating new beta cell formation; a 3-fold rise intotal beta cells staining insulin in STZ-rendered diabetic mice wasobserved. Levetan C S., et al, Endocr Pract. 2008; 14(9):1075-1083. Thehuman Reg3a protein and placebo-treated mice underwent an overnight fastand a fasting glucose level on the morning of day 39 of treatment.Fasting glucose levels were 258.00±84.5 mg/dl in the placebo groupcompared to a fasting glucose level of 111.00±11.4 mg/dL (P=0.020) inthe Reg3a protein-treated mice.

Two studies by separate investigators have shown the ability of Regpeptide to transform human extra-islet pancreatic exocrine tissue intonew beta cells in vitro. These studies were conducted by a methodologyutilized in pancreatic islet transplantation in which the pancreaticendocrine beta cells are separated from the exocrine ductal tissue; theexocrine ductal tissue was shown to transform into new beta cells in thepresence of hamster Reg3gamma peptide. Li J, et al. Peptides 2009;30:2242-9, Assouline-Thomas B G, Diabètes 2008, 57(Suppl; 1) A2413. Thecurrent gold-standard, BrdU labeling, was used to label the beta celllineage in rodents, which distinguishes whether new beta cells areformed by budding from pre-existing beta cells versus being formed fromextra-islet ductal exocrine tissue. Kapur R, et al, Islets. 2012; 4(1).

The Section of Islet Cell and Regenerative Biology at Joslin DiabetesCenter found that the 15-amino acid hamster Reg3 gamma peptide waspresent in the newest beta cells and islets that were formed directlyfrom branching proliferating extra-islet ducts, which also confirms thatthe mechanism of action of Reg peptide is to form new beta cells fromextra-islet exocrine tissue. Guo L et al, Diabetes. 2010, 59(suppl; 1)A2589. When Reg is inhibited by the administration of a blockingantibody in an animal model of pancreatic injury there was attenuatedrecovery, also confirming that Reg's role is both protective andregenerative during acute pancreatic injury. Viterbo D, et al. JOP.2009; 10(1):15-23.

The Departments of Beta Cell Regeneration at the Hagedorn ResearchInstitute and Peptide and Protein Chemistry at Novo Nordisk reported a2-fold increase in the volume of new small islets developing fromnon-endocrine tissue resulting from the treatment with both the human 14amino acid Reg3a peptide, HIP, and the 15-amino acid Reg3gamma hamsterpeptide Kapur R, et al, Islets. 2012; 4(1). Five days after treatmentwith both the 14-amino acid human Reg3a peptide, HIP, and the 15-aminoacid hamster Reg3gamma peptide, INGAP, there were increased levels ofnew islet markers necessary for islet formation, including NGN3, NKX6.1,SOX9, and INS, indicating that REG is a catalyst for beta cellneogenesis. Kapur R, et al, Islets. 2012; 4(1). Similar to thesefindings, other data support that the human Reg protein and the hamsterReg3gamma peptide are an initiating factor for downstream regulation ofnew beta cells. Levetan C., 2010, J Diabetes; 2(2):76-84. For example,when Reg is initially expressed, PDX-1, PAX1, Ngn3, Nkx6.1, Sox9, andIns are not expressed; once Reg is present, PDX-1, PAX1, Ngn3, Nkx6.1,Sox9 and Ins and other beta cell proliferation factors become presentdemonstrating that Reg activates downstream factors necessary for betacell regeneration. Vukkadapu S S Physiol Genomics 2005:21, 201-211,Kapur R., et al., Islets. 2012; 4(1):Epub. Gun and colleagues confirmedpositive Reg staining in ductal epithelium in acutely diabetic NOD miceand in the pancreas of a type 1 healthy cadaveric human pancreata or inhealthy mice.

The organ specificity of the hamster Reg3 gamma protein to thepancreatic ducts has been illustrated by the tagged Reg3gamma hamsterprotein labeled with fluorescein isothiocyanate that was administeredvia intraperitoneal injection to rodents. The only organ that hadfluorescent staining was the pancreas with labeling only foundspecifically to be within the nonendocrine pancreatic ductalpopulations, again confirming that the mechanism of action of Reg istransformation of extra-islet ductal cells into beta cells. Pittenger GL et al, Diabetologia 2009; 52 (5):735-738. There are now numerousstudies confirming that the mechanism of action of the Reg peptides isto transform extra-islet exocrine ductal tissue into new islets ratherthan the newly formed beta cells resulting from the budding fromexisting beta cells.

This inventor has also investigated the role and pathways of other humanhormones involved in beta cell regeneration with findings consistentwith initial findings of Moore and colleagues in 1906, demonstrating therole of gastrointestinal hormones in improving diabetes control amongthree patients with type 1 diabetes. Levetan C. 2010, J Diabetes;2(2):76-84, Moore et al, Biochem J. 1906; 1(1): 28-38. The mechanism ofaction of these gastrointestinal hormones were not only found to be ininsulin secretion, but decades later these gut peptides have been shownto be involved in the transformation of extra-islet exocrine tissue intonew endocrine tissue containing beta cells. Wang T C. J Clin Invest.1993; 92(3):1349-56.

Not until 1999, when the use of cell lineage labeling became available,did the embryological concepts of the pancreas change. Whereas it hadbeen thought that the pancreas was derived from both ectoderm andendoderm, it has now been shown that the entire pancreas arises onlyfrom endoderm during embryological development. This helps explain howbeta progenitor cells have been described as residing diffuselythroughout the adult pancreatic tissue and how growth factors transformpancreatic extra-islet ductal tissue into new beta cells. Over the pastseveral decades, the ability to regenerate new beta cells fromprogenitor cells found within the pancreatic ductal tissue has beenillustrated by many teams.

Despite some promise using the hamster Reg3gamma peptide in patientswith type 1 diabetes with a 27% rise in stimulated c-peptide among type1 patients with no detectable c-peptide at baseline, without 1) atolerable agent that patients are able to use and 2) the usage of animmune tolerance agent combined with any such agent that can increasebeta regeneration, an improved impact on insulin requirements is notlikely to be sustained in trials (Lipsett M. Cell Biochem Biophys. 2007;48(2-3):127-3). Data from J J Meier and colleagues demonstrates that thenewest beta cells are the ones that are most vulnerable tocytokine-induced death and trigger autoimmune attack. Meier J J et alDiabetologia 2006; 49(1):83-9.

Despite early findings of patients with type 1 diabetes demonstrating asignificant reduction in insulin requirements and improvements instimulated c-peptide within 54 days of usage among type 1 patients,sustained results have not been seen and very poor tolerability withmore than ⅓ of patients receiving such severe skin site reactions not tocontinue in the trial, further clinical development of optimizedversions of the 15 amino acid hamster Reg3gamma peptide have beenabandoned. This inventor discloses an optimized 15 amino acid hamster3gamma peptide that can be further used in combination with an immunetolerance agent to protect newly formed beta cells from autoimmunedestruction, for sustained insulin independence.

Some success has temporarily been seen among immune tolerance agentsutilized among recent onset type 1 patients, but without new beta cellformation over time, the limited amount (fewer than 10%) of beta cellsremaining at the time of type 1 diagnosis will undergo apoptosis untilpatients require insulin again. Clinical trials using hamster Reg3 gammaalone have concluded that 1) lack of tolerance due to injection siteswelling and pain due to the large amount of peptide required 2) lack ofsustained efficacy 3) lack of insulin independence have not led toconsideration of new formulations of hamster Reg3 gamma that may be moretolerable in man and allowing for lower dosages.

Thus, for hamster Reg3gamma to be successful, both a new formulationthat allows for more sustained action with lower amounts of drug arecritical for potential success in man that has been seen in animalmodels. Additionally, regeneration agents alone such as with proton pumpinhibitors (lansoprazole) and DPP-4 inhibitors (sitagliptin), which haveshown success in mouse models do not reflect the lack of success in manamong newly diagnosed type 1 patients who have shown neither long terminsulin independence with a regeneration agent or an immune toleranceagent. Certain aspects of the invention, require that the optimizedhamster Reg3gamma peptide be used with an immune tolerance agent amongtype 1 patients and to have the immune tolerance agent on board at thetime that new beta cells are being generated.

The immunosuppressive drug Cyclosporine has been shown to have long-termsafety and short-term efficacy for rendering new onset patients withtype 1 diabetes insulin-independent. The immunosuppressive effects ofCyclosporine were discovered in 1972 in a screening test on immunesuppression designed and implemented by Dr. Hartmann Stateline. Thesuccess of Cyclosporine in preventing organ rejection was later shown inkidney transplants by Calne and colleagues at the University ofCambridge and in liver transplants performed initially at the Universityof Pittsburgh Hospital. Cyclosporine was subsequently approved for usein 1983. Since then, it has been used to prevent and treatgraft-versus-host reactions in bone marrow transplantation and toprevent rejection of kidney, heart, and liver transplantation.

In addition to transplants, Cyclosporine has also been used inpsoriasis, severe atopic dermatitis, pyoderma gangrenosum, chronicautoimmune urticaria, and, infrequently, in rheumatoid arthritis andrelated diseases. It is commonly prescribed in the US as an ophthalmicemulsion for the treatment of dry eyes. Cyclosporine has also been usedto help treat patients with acute severe ulcerative colitis that do notrespond to treatment with steroids. This drug is also used as atreatment of posterior or intermediate uveitis with noninfectiveetiology. Cyclosporine is also currently used to experimentally treatcardiac hypertrophy.

Twenty-five years ago, Bougneres and colleagues reported in the NewEngland Journal of Medicine that among forty children between the agesof 7 and 15 years of age with recent onset type 1 diabetes, 67.5% ofpatients were able to discontinue insulin within 48+5 days of initiationof 7.5 mg/kg/day of Cyclosporine in two divided dosages. Bourgneres P F.N Engl J Med 1988; 318:663-670). By 12 months after the initiation ofCyclosporine, 50% of patients remained insulin free. Over the next sixyears of follow-up the initial cohort was pooled with 43 more childrenwith recent onset type 1 diabetes for a total of 83 children givenCyclosporine, who were compared to 47 children with new onset type 1diabetes during the same time period who were not treated withCyclosporine. DiFillippo G Diabetes 45:101-104, 1996. Over the first 4years, the Cyclosporine-treated group kept plasma C-peptide at levelstwice as high as the control group (P<0.02). It took 5.8 years forglucagon-stimulated C-peptide to become undetectable in the Cyclosporinegroup vs. 3.2 years in the control group. Average insulin dose remainedlower by 0.2-0.4 units/kg/day. Hemoglobin A1C was lower by 1% in theCyclosporine-treated group who also had significantly less hypoglycemiathan the diabetic control subjects (P<0.05). After four years, thedifferences between the groups became non-significant. Other studieshave found similar data that Cyclosporine had a positive impact onrecent onset type 1 diabetes patients, but over time, all patientsrequired insulin. (The Canadian-European Randomized Control Trial Group.Diabetes 1988; 37:1574-82, Assan R. Diabetes Metab Res Rev 2002;18:464-472, Feutren G. Lancet. 1986 Jul. 19; 2(8499):119-24). In onetrial of 285 patients with recent onset type 1 diabetes whom weretreated for a mean of 20 months with 7.5 mg/kg/day of Cyclosporine,there were permanent renal side effects seen after following patientsfor 13 years. Patients in this study received renal biopsies withextensive non-invasive renal follow-up. Even patients with moderatekidney lesions on biopsy at 1 year had normal and stable clearancevalues at 7 to 13 years (Assan R. Diabetes Metab Res Rev 2002;18:464-472).

Trials with Cyclosporine fell out of favor because there were nopermanent remissions over time. Lack of permanent remission ishypothesized by this inventor to be due to the data suggesting that asin the case of Cyclosporine and more than a dozen other agents over thepast decade utilized for protecting the beta cells from furtherautoimmune attack, the agents are unable to impact the remaining betacells in the pancreas. It is estimated that fewer than 10% offunctioning beta cells remain at the time of diagnosis of type 1diabetes. Despite trials showing a positive impact of many autoimmunetherapies initiated within twelve weeks of symptoms and diagnosis oftype 1 diabetes, none have resulted in lasting insulin independence.Immune tolerance agents utilized among recent onset type 1 patients thatin addition to Cyclosporine have shown a potential immune benefit buthave not resulted in significant or sustained insulin independenceinclude, but are not limited to the heat shock protein 60, Diapep 277,Bacille Calmette-Guérin (also known as the BCG vaccine and commonlyknown as the vaccine against tuberculosis), mycophenolate mofetil,daclizumab, rituximab (anti CD20), anti CD3 antibodies including hOKT3gamma1 (Ala-Ala), and the monoclonal antibody TRX4 (ChAglyCD3), CTLA4-Ig(abatacept) a selective co-stimulation modulator as it inhibits theco-stimulation of T cells, campath-1H, anti-CD52 antibody, a humanizedmonoclonal antibody to T-cells, polyclonal anti-T-lymphocyte globulin(ATG), GAD antibody vaccine based on the 65 kDa isoform of therecombinant human glutamic acid decarboxylase protein (rhGAD65),diazoxide and Alpha-1 Antitrypsin.

Type 2 diabetes results from a different etiology, but similar to type 1diabetes there is a substantial loss of 50-75% of beta cell mass at thetime of diagnosis; however, the loss is not as acute as that seen fromthe autoimmune destruction in type 1 diabetes. The beta cell loss seenin type 2 diabetes is due to a more chronic beta cell loss that isimpacted by a number of factors including lifestyle, free fatty acidsand genetics. Thus, while the beta call loss is not due to suddenautoimmune destruction, there is still the need for beta cellregeneration and sustained beta cell mass.

To date, there have been no studies that combine an immune toleranceagent with a known beta cell regeneration growth factor that has beenshown to directly stimulate the formation of new beta cells from ductalcells. One of the reasons that this combination of an immune toleranceagent with a Reg peptide is that it has not previously been consideredand has not been obvious is because dozens of preclinical trials withrodent type 1 diabetes models including NOD mouse models have shown onlythe need for gastrin and other beta cell growth factors for reversal ofdiabetes. Likewise, rodent type 1 diabetes models including NOD mousemodels have shown that using an immune tolerance agents alone is allthat is needed to reverse type 1 diabetes in mice.

This inventor has shown great distinctions between the insulin-producingislets of mice and men with humans having much more complex isletstructures with respect to composition of cell type, neural and vascularinnervation and unique paracrine interactions that are not found inrodents. Levetan has demonstrated vast differences in the islets of miceand men, which may explain the many, many studies conducted among rodentmodels in the field of diabetes that later are unable to be replicatedin human studies. Levetan C S et al. Endocr Pract. 2012; 27:1-36. [Epubahead of print]. Specifically, trials with multiple different agents andtypes of agents have been utilized in preclinical rodent modelsevaluating agents that may be successful in clinical practice for usagein patients with type 1 diabetes. This inventor has also previouslyshown, like many other scientific teams, that after fetal development ofbeta cells, typically new beta cells are only derived from the existing,surviving beta cell population. Different and unique to the previous artin the field, this inventor has shown the ability to post-natallygenerate new beta cells by the transformation of human pancreatic ductaltissue. Levetan C. J Diabetes. 2010; 2(2):76-84, Levetan C S. EndocrPract. 2008; 14(9):1075-83.

New and unique research by this inventor, which has not been obvious inthe prior art, is 1) the ability to reverse diabetes in the diabeticmouse models may be flawed by the complexity of the human islet comparedto that of the rodent and 2) the process of generating new beta cellsmust be from a different source than from the beta cells remaining afterthe diagnosis of type 1 or type 2 diabetes is made because of thelimited supply (<10% for type 1 diabetes and <50-75% for type 2diabetes). This inventor has shown the ability to transform new pools ofbeta cells within new islets from extra-islet ductal tissue (See U.S.Pat. Nos. 8,211,430, 7,989,415, 7,714,103 and 7,393,919).

There is a need in the art for new therapeutic modalities for thetreatment of diabetes in humans that generate new beta cells fromextra-islet tissue while preserving the population of nascent beta cellsfrom destruction by the immune system.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the present invention provide for novel therapies,pharmaceutical compositions and methods for insulin independenceutilizing a new optimized hamster Reg3 gamma peptide, which is new tothe art and has not previously been considered for development in the 30year history since its discovery. Methods, pharmaceutical compositionsand therapies novel to the prior art are utilized in this invention torender patients with recent onset and existing type 1 diabetes insulinindependent by an optimized hamster Reg3 gamma peptide and an immunetolerance agent for type 1 patients to become insulin independent andused alone without an immune tolerance agent for type 2 diabetes. Whilenot wishing to be bound by theory, optimized Reg3 gamma peptidesincreases beta cell generation by its demonstrated properties shownwithin of transforming ductal pancreatic cells into new islets. Newislets contain new populations of not only beta cells generating insulinand amylin, but also, alpha cells making glucagon, delta cells makingsomatostatin, epsilon cells making islet ghrelin and gamma cells makingpancreatic polypeptide, all of which are necessary for glucosehomeostasis.

The ability to use a new Reg peptide agent that is tolerated in man togenerate new islets from one's one pancreatic ductal tissue, provides acompletely new approach to the treatment of diabetes. This inventionidentifies a new utilization of a peptide therapy, which has previouslynot been tolerated in man because the amount of peptide necessary togive optimal results has not been able to be given because of local sideeffects, which have limited its use and development over 30 years sinceits discovery.

This invention identifies for the first time the use of an optimizedform of hamster Reg3 gamma peptide that 1) is tolerated and will not belimited in usage by local trauma due to the large dosage required 2)when combination of usage of this optimized hamster Reg3gamma peptidewith an immune tolerance agent for the protection of the new beta cellsprovides for the potential for insulin independence among type 1patients 3) provides therapy for the underlying cause of both type 1 and2 diabetes by the transformation of ductal progenitor cells into new5-celled fully functional islets 4) provides innovative therapy for type1 and 2 diabetes and many conditions of insulin deficiency.

The therapeutic methods described in this invention are not containedwithin the prior art, and specifically include, but are not limited tothe usage of a specific optimized 15 amino acid hamster Reg3 gammasequence in combination with or without an immune tolerance agent, butinclude methods for usage in type 1 and 2 diabetes and conditions ofinsulin deficiency. Specifically the high dosages required of thishamster Reg3 gamma 15 amino-acid hamster peptide have resulted in severelocal discomfort and swelling in the area of the subcutaneous injectionlimiting the success and usage of a peptide which has shown the abilityto increase endogenous insulin production by 27% by day 54 in type 1patients with no baseline c-peptide, yet, optimization of the peptidesince its first clinical usage 20 years ago, has not been considered.Clinical trials have failed to develop a tolerable therapy despite clearin vitro mechanisms of action showing the formation of new beta cellformation from human pancreatic ductal tissue. The potential for this 15amino-acid hamster Reg3 gamma peptide is enormous once it is optimizedand made tolerable for usage in man. Despite its discovery in 1983, anoptimized hamster Reg3 gamma peptide has never been formed, considered,invented or utilized prior, nor has an optimized hamster Reg3 gammapeptide been considered for usage with an immune tolerance agent, whichthis invention demonstrates is required to protect new beta cells formedby the optimized hamster Reg3 gamma peptide, from autoimmunedestruction.

This invention also provides for novel therapies, pharmaceuticalcompositions and methods for insulin independence among type 2 diabetespatients using an optimized hamster Reg3 gamma peptide alone or incombination with other therapies as in the case of both type 1 and 2diabetes. The development of an optimized Reg3 gamma hamster peptide hasnever previously been proposed or utilized in clinical trials in type 1or two diabetes. One of the reasons that this combination of an immunetolerance agent with the hamster Reg3 gamma peptide has been consideredenough in NOD trials, but this has not been the case in humans and thehamster Reg3 gamma has been so poorly tolerated that efficacious dosageshave not been delivered to render patients insulin free.

Because dozens of preclinical trials with rodent type 1 diabetes modelsincluding NOD mouse models have shown only the need for gastrin andother beta cell growth factors for reversal of diabetes. Likewise,rodent type 1 diabetes models including NOD mouse models have shown thatusing an immune tolerance agents alone is all that is needed to reversetype 1 diabetes in mice. This inventor has shown great distinctionsbetween the insulin-producing islets of mice and men with humans havingmuch more complex islet structures with respect to composition of celltype, neural and vascular innervation and unique paracrine interactionsthat are not found in rodents.

This invention provides a new model for treatment of type 1 and 2diabetes. Based upon the complexity and distinctions between the isletsof mice and men, this invention provides for a novel therapy consistingoptimization of hamster Reg3 gamma peptide with pharmaceuticalcompositions and methods for insulin independence and provides amethodology for treating patients requiring insulin that have notpreviously been described. Embodiments of the present invention provideformulations, derivatives, and modifications for the purpose ofoptimizing a 15 amino-acid peptide bioactive region on the hamster Reg3gamma gene protein that has unique homology in sequence to other humanReg proteins, yet, no formulations, derivatives and modifications ofthis peptide have been made for it to be tolerable for usage in man.This disclosure provides specific methods for usage, including optimizedversions of hamster Reg3 gamma peptides that would enable usage in man.In contrast, native hamster peptide has been found since its discoveryto have too limited stability, and even when used in man in dosages thatwere not great enough to see significant impact on patients withdiabetes, patients enrolled in clinical trials had such significantlocal side effects that ⅓ of patients with type 1 diabetes dropped outof clinical trials due to pain, bruising and infection to the localinjection site of the hamster Reg3 gamma 15 amino acid peptide. Thisinvention provides for a novel look at this peptide and provides for thecapability of the hamster Reg3 gamma peptide to be used in man viageneration of a more tolerable, stable and effective therapy that can beused for 1) the regeneration of beta cells among patients with diabetes.Methods of using this optimized hamster Reg3 gamma 15-amino acidpeptide, analogs and nonpeptide peptidomemetics for treatment andreversal of type 1 diabetes and type 2 diabetes and conditions ofinsulin deficiency are provided.

In one embodiment, the optimized 15 amino acid hamster Reg3 gammapeptide aminoIle-Gly-Leu-His-Asp-Pro-Ser-His-Gly-Thr-Leu-Pro-Asn-Gly-Ser (SEQ IDNO: 1) and derivatives thereof are designated as agents for thetreatment for 1) reversal of type 1 by enhancing beta cell replicationwhen utilized in conjunction with an immune tolerance agent and for type2 diabetes when utilized alone or in combination with lifestylemanagement and/or in combination with other diabetes agents byembodiments that allow for greater tolerability and stability in man,thus enabling the 15 amino acid hamster Reg3 gamma peptide to become atherapeutic in man for usage for the treatment of diabetes and insulindeficient states.

This peptide may also be represented by the formula: T; wherein T=SEQ IDNO:1. In another embodiment, the 15 amino sequence hamster Reg3 gammapeptide (SEQ ID NO:1) is optimized by capping the ends. Optimization ofthe SEQ ID NO: 1 and to improve the Tmax and bioavailability of thispeptide includes, but is not limited to blocking with an n-terminalacetyl group and a c-terminal amide group as provided below:

(SEQ ID NO: 2) Ac--Ile-Gly-Leu-His-Asp-Pro-Ser-His-Gly-Thr-Leu-Pro-Asn-Gly-Ser-NH₂.

This peptide may also be represented the formula: Ac-T-NH₂, where T=SEQID NO:1, Ac=Acetyl group, and NH₂=Amide group.

SEQ ID NO: 3 is the 15 amino acid hamster Reg3 gamma peptide which hasan additional n-terminal cysteine residue, in purified, synthetic orrecombinant form:

(SEQ ID NO: 3) Cys-Ile-Gly-Leu-His-Asp-Pro-Ser-His-Gly-Thr-Leu-Pro-Asn-Gly-Ser 

This peptide may be represented by Cys-T or U; wherein T=SEQ ID NO:1,Cys=Cysteine, and U=SEQ ID NO:3.

In another embodiment, a dimer of the peptides represented by SEQ IDNO:3 is provided as SEQ ID NO:4, in purified, synthetic, or recombinantform. The dimer forms via the creation of a disulfide bond between thecysteine residues of the individual monomers:

(SEQ ID NO: 4)Cys-Ile-Gly-Leu-His-Asp-Pro-Ser-His-Gly-Thr-Leu-Pro-Asn-Gly-Ser  |Cys-Ile-Gly-Leu-His-Asp-Pro-Ser-His-Gly-Thr-Leu-Pro-Asn-Gly-Ser. 

This peptide may be represented by

wherein T=SEQ ID NO:1, Cys=Cysteine, U=SEQ ID NO:3, and dashed linerepresents a disulfide bond.

In another embodiment, a dimer of the peptides represented by SEQ IDNO:3 is provided as SEQ ID NO:5, wherein each monomer has been modifiedto include an n-terminal cysteine residue and has been blocked with an-terminal acetyl group and an c-terminal amide group, in purified,synthetic, or recombinant form. The dimer forms via the creation of adisulfide bond between the cysteine residues of the individual monomers:

(SEQ ID NO: 5)Ac-Cys-Ile-Gly-Leu-His-Asp-Pro-Ser-His-Gly-Thr-Leu-Pro-Asn-Gly-Ser-NH₂    |Ac-Cys-Ile-Gly-Leu-His-Asp-Pro-Ser-His-Gly-Thr-Leu-Pro-Asn-Gly-Ser-NH₂.

This peptide may be represented by

wherein T=SEQ ID NO:1, Cys=Cysteine, U=SEQ ID NO:3, Ac=Acetyl group,NH₂=Amide group, and dashed line represents a disulfide bond.

In another embodiment, the peptide of SEQ ID NO:2 has been covalentlybonded to a dimeric maleimide activated 40 Kd PEG construct, inpurified, synthetic, or recombinant form, and is provided as SEQ IDNO:6:

(SEQ ID NO: 6) (PEG)₂-Cys-Ile-Gly-Leu-His-Asp-Pro-Ser-His-Gly-Thr-Leu-Pro-Asn-Gly-Ser.

This peptide may be represented by (R)₂-Cys-T or (R)₂-U;

wherein R=PEG, Cys=Cysteine, T=SEQ ID NO:1, and U=SEQ ID NO:3

In another embodiment, the peptide of SEQ ID NO:2 is blocked with ann-terminal acetyl group and a c-terminal amide group, and the n-terminalcysteine residue has been covalently bonded to a dimeric maleimideactivated 40 Kd PEG construct, in purified, synthetic, or recombinantform, and is provided as SEQ ID NO:7:

(SEQ ID NO: 7)Ac-Cys-Ile-Gly-Leu-His-Asp-Pro-Ser-His-Gly-Thr-Leu-Pro-Asn-Gly-Ser-NH₂    |   (PEG)₂. 

This peptide may be represented by

wherein R=PEG, Cys=Cysteine, T=SEQ ID NO:1, U=SEQ ID NO:3, Ac=Acetylgroup, and NH₂=Amide group

Such modifications render the sequence less susceptible to proteasecleavage in serum with proteases that normally recognize free ends,thereby effectively increasing the Tmax and bioavailability of thepeptides. Peptides modified in this manner demonstrate increasedefficacy thereby requiring decreased dosages when administered by forexample, IV, IM, SubQ or intraperitoneal routes.

In various embodiments, the SEQ ID NOS:1-7 may be provided alone orformulated in a pharmaceutical composition alone or in combination withan immune tolerance agent such as Cyclosporine in a pharmaceuticallyacceptable carrier. Furthermore, this disclosure provides additionalstructures beyond those described in SEQ ID NOS:1-7 such that anoptimized Reg3 gamma peptide comprising any combination of addition ofan N-terminal cysteine, acetylation at the N-terminus, amidated at theC-terminus, and/or PEGylated at either terminus, in monomeric or dimericform, is provided. These additional structures may be provided alone orformulated in a pharmaceutical composition alone or in combination withan immune tolerance agent such as Cyclosporine in a pharmaceuticallyacceptable carrier.

An exemplary embodiment provides a pharmaceutical composition comprisinga peptide comprising SEQ ID NO:1 and an immune tolerance agent such asCyclosporine.

Another exemplary embodiment comprises an optimized Reg3 gamma peptidecomprising the sequence of SEQ ID NO:1 that is acetylated at theN-terminus, amidated at the C-terminus, and/or PEGylated at eitherterminus. The optimized Reg 3 gamma may have the N-terminus orC-terminus covalently bonded to a dimeric maleimide activated 40 Kd PEGconstruct. The optimized Reg3 gamma peptide may be provided alone orformulated in a composition alone or in combination with an immunetolerance agent such as Cyclosporine in a pharmaceutically acceptablecarrier.

Another exemplary embodiment comprises an optimized Reg 3 gamma peptidecomprising the sequence of SEQ ID NO:3 that is acetylated at theN-terminus, amidated at the C-terminus, and/or PEGylated at eitherterminus. The optimized Reg 3 gamma peptide may have the N-terminus orC-terminus covalently bonded to a dimeric maleimide activated 40 Kd PEGconstruct. The optimized Reg3 gamma peptide may be provided alone orformulated in a composition alone or in combination with an immunetolerance agent such as Cyclosporine in a pharmaceutically acceptablecarrier.

Another exemplary embodiment comprises an optimized Reg 3 gamma peptidewhich is an isolated peptide homodimer, wherein each molecule consistsof the amino acid sequence of SEQ ID NO:3 and the homodimer is obtainedby dimerization through the free cysteine in the two molecules of thedimer. The optimized Reg 3 gamma peptide homodimer may be acetylated atthe N-terminus, amidated at the C-terminus, and/or PEGylated at eitherterminus. The optimized Reg3 gamma peptide may have the N-terminus orC-terminus covalently bonded to a dimeric maleimide activated 40 Kd PEGconstruct. The optimized Reg3 gamma peptide homodimer may be providedalone or formulated in a composition alone or in combination with animmune tolerance agent such as Cyclosporine in a pharmaceuticallyacceptable carrier.

Another exemplary embodiment comprises a method of treating type 1 ortype 2 diabetes, comprising administering to a subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a peptidecomprising SEQ ID NO:1 and an immune tolerance agent such asCyclosporine.

Another exemplary embodiment comprises a method of treating type 1 ortype 2 diabetes, comprising administering to a subject a therapeuticallyeffective amount of a pharmaceutical composition comprising anyoptimized Reg3 gamma peptide of this disclosure alone or in combinationwith an immune tolerance agent such as Cyclosporine.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION

All references listed in this specification, including books, journalarticles, published patent applications, and issued patents, are herebyincorporated by reference in their entirety.

To date, over the past thirty years, the hamster Reg3 gamma peptide hasnot been able to be utilized by patients with diabetes due to poorstability and tolerability in man, despite the safe and efficaciouspreclinical and early clinical results from decades ago. Throughout thehistory of the 15 amino acid hamster Reg3gamma peptide and itsintroduction to human patients with type 1 and 2 diabetes, it has notbeen developed nor have there been any changes in formulation to make ita potential therapeutic among patients with diabetes.

In one embodiment, derivatives of hamster Reg3 gamma peptide are createdby blocking the peptide with an N-terminal acetyl group and a C-terminalamide group to form the peptide of SEQ ID NO: 2. In another embodiment,derivatives of hamster Reg3 gamma peptide are created by adding acysteine residue to the n-terminal of SEQ ID NO: 1 to form Hamster Reg3gamma Cys (SEQ ID NO: 3) which results in a compound which is capable offorming dimers in solution to form HamsterReg3 gamma CysDimer (SEQ IDNO: 4). Such a modification increases the stability hamster Reg3gammaCys variants by avoiding proteases which recognize hamster Reg3gamma Cysor hamster Reg3gamma Cys variants in monomer form.

In another embodiment, hamster Reg3 gamma Cys variants are blocked withan N-terminal acetyl group and a C-terminal amide group. Suchmodifications render the sequence less susceptible to protease cleavagein serum with proteases that normally recognize free ends and resultingin a compound which is capable of forming dimers in solution hamsterReg3 gamma Cys Blocked Dimer (SEQ ID NO: 5), thereby increasing thestability of hamster Reg3 gamma peptide Cys Blocked variants by avoidingproteases which recognize hamster Reg3 gamma or Hamster Reg3 gammapeptide Cys Blocked variants in monomer form.

In another embodiment, hamster Reg 3gamma peptide Cys variants aremodified by covalently binding a dimeric maleimide activated 40 Kd PEGconstruct to the n-terminal cysteine residue hamster Reg3 gamma peptideCysPEG (SEQ ID NO: 6). Such a modification improves the stability ofhamster Reg3gammaCys in serum resulting in increased bioavailability anddosing efficacy of hamster Reg3gamma are variants in therapeuticstrategies for beta cell replication for reversing diabetes in vivo.

In another embodiment, hamster Reg3 gamma CysBlocked variants aremodified by covalently binding a dimeric maleimide activated 40 Kd PEGconstruct (Hamster Reg3gamma peptide CysBlockedPEG (SEQ ID NO: 7). Sucha modification improves the stability hamsterReg3 gamma CysBlockedvariants in serum resulting in increased bioavailability and dosingefficacy of hamster Reg3gamma peptide CysBlocked variants in therapeuticstrategies for stimulating islet neogenesis and reversing diabetes invivo.

In another embodiment, a hamster Reg3 gamma peptide analogs and hamsterReg3 gamma peptidomemetics are designed to improve the efficacy andtherapeutic ability for beta cell replication for improving andreversing diabetes in vivo.

Further embodiments of the present invention provide methods foradministering the optimized hamster Reg3gamma peptide compounds (SEQ IDNOs: 1-7) alone or in combination with other therapeutic agents forstimulating pancreatic islet cell regeneration. In various embodiments,the methods of the invention can be practiced by administration of atherapeutically effective amount of optimized hamster Reg3 gamma peptidealone, in combination with insulin, in combination with insulin andanother agent, and in combination with one or more agents other thaninsulin.

The peptides may be produced through recombinant molecular biologytechniques or solid phase synthesis techniques. Recombinant molecularbiology techniques include those described in Molecular Cloning: ALaboratory Manual, Green and Sanbrook, 2012. Solid-phase synthesistechniques are described in Merrifield, in J. Am. Chem. Soc.,15:2149-2154 (1963), M. Bodanszky et al., (1976) Peptide Synthesis, JohnWiley & Sons, 2d Ed.; Kent and Clark-Lewis in Synthetic Peptides inBiology and Medicine, p. 295-358, eds. Alitalo, K., et al. SciencePublishers, (Amsterdam, 1985); as well as other reference works known tothose skilled in the art such. A summary of peptide synthesis techniquesmay be found in J. Stuart and J. D. Young, Solid Phase PeptideSynthelia, Pierce Chemical Company, Rockford, Ill. (1984), which isincorporated herein by reference. The synthesis of peptides by solutionmethods may also be used, as described in The Proteins, Vol. II, 3d Ed.,p. 105-237, Neurath, H. et al., Eds., Academic Press, New York, N.Y.(1976). Appropriate protective groups for use in such syntheses will befound in the above texts, as well as in J. F. W. McOmie, ProtectiveGroups in Organic Chemistry, Plenum Press, New York, N.Y. (1973), whichis incorporated herein by reference. In general, these synthetic methodsinvolve the sequential addition of one or more amino acid residues orprotected amino acid residues to a growing peptide chain. Normally,either the amino or carboxyl group of the first amino acid residue isprotected by a suitable, selectively removable protecting group. Adifferent, selectively removable protecting group is utilized for aminoacids containing a reactive side group, such as lysine. Block synthesistechniques may also be applied to both the solid phase and solutionmethods of peptide synthesis. Rather than sequential addition of singleamino acid residues, preformed blocks comprising two or more amino acidresidues in sequence are used as either starting subunits orsubsequently added units rather than single amino acid residues.Alternative or additional peptide synthesis methods and techniques canbe found in Peptide Chemistry: A Practical Textbook: 2nd Edition, MiklosBodanszky, 1993.

Proteins having the amino acid sequence of SEQ ID NOS:1-7 thereof or aportion thereof may also be synthesized by solid-phase peptide synthesisusing procedures similar to those described by Merrifield, 1963, J. Am.Chem. Soc., 85:2149. During synthesis, N-a-protected amino acids havingprotected side chains are added stepwise to a growing polypeptide chainlinked by its C-terminal and to an insoluble polymeric support, i.e.,polystyrene beads. The proteins are synthesized by linking an aminogroup of an N-a-deprotected amino acid to an a-carboxyl group of anN-a-protected amino acid that has been activated by reacting it with areagent such as dicyclohexylcarbodiimide. The attachment of a free aminogroup to the activated carboxyl leads to peptide bond formation. Themost commonly used N-a-protecting groups include Boc, which is acidlabile, and Fmoc, which is base labile. Details of appropriatechemistries, resins, protecting groups, protected amino acids andreagents are well known in the art and so are not discussed in detailherein (See, Atherton et al., 1989, Solid Phase Peptide Synthesis: APractical Approach, IRL Press, and Bodanszky, 1993, Peptide Chemistry, APractical Textbook, 2nd Ed., Springer-Verlag).

Purification of the resulting optimized is accomplished usingconventional procedures, such as preparative HPLC using gel permeation,partition and/or ion exchange chromatography. The choice of appropriatematrices and buffers are well known in the art and so are not describedin detail herein.

Protocols for blocking peptides with acetyl and amide groups are knownin the art and can be found in a number of protein protocol textbooksknown in the art. Specific examples include those published in Methodsin Molecular Biology, Vol. 35: Peptide Synthesis Protocols, Chapter 8:Site-Specific Chemical Modification Procedures, Edited by M W Penningtonand B M Dunn, 1994, as well as U.S. Pat. No. 4,708,934, U.S. Pat. No.5,503,989, U.S. Patent Application Publication No. US 20060127995.Alternative or additional blocking procedures can be found in PeptideChemistry: A Practical Textbook: 2nd Edition, Miklos Bodanszky, 1993.

Inert polymer molecules such as high molecular weight polyethyleneglycol(PEG) can be attached to a peptide of this disclosure or an analog orderivative thereof with or without a multifunctional linker eitherthrough site-specific conjugation of the PEG to the N- or C-terminus ofthe protein or via epsilon-amino groups present on lysine residues.Linear or branched polymer derivatization that results in minimal lossof biological activity can be used. The degree of conjugation can beclosely monitored by SDS-PAGE and mass spectrometry to ensure properconjugation of PEG molecules. Unreacted PEG can be separated frompeptide-PEG conjugates by size-exclusion or by ion-exchangechromatography.

The optimized peptides may also be PEGylated at cysteine residuesthrough maleimide chemistry. Maleimide-activated PEG reacts with thethiols of cysteine residues of protein and to form stable thioetherlinkages and are highly stable against hydrolysis. The maleimide moietyreacts rapidly with the thiol group without hydrolysis around neutralpH. Protocols for creating maleimide-activated PEG constructs may befound in Schumacher et al., In Situ Maleimide Bridging of Disulfides anda New Approach to Protein PEGylation, Bioconjugate Chem., 2011, 22 (2),pp 132-136, Doherty et al., Site-Specific PEGylation of EngineeredCysteine Analogs of Recombinant Human Granulocyte-MacrophageColony-Stimulating Factor, Bioconjug Chem. 2005; 16(5): 1291-1298, USPatent Application Publication No. 20090298746 A1, European Patent No.EP 1881850 B1, European Patent No. EP 2178900 B1.

Other embodiments of the present invention provide pharmaceuticalformulations and unit dose forms of optimized hamster Reg3gamma peptide.In one embodiment, the pharmaceutical formulation provided containsoptimized hamster Reg3 gamma peptide alone or in combination with one ormore other active pharmaceutical ingredients (APIs). In one embodiment,the API is an or agents in soluble liposome preparations that allow theoptimized hamster Reg3gamma peptide to be administered by a variety ofroutes, including subcutaneously, intramuscularly, intravenously, andeven orally, depending on the formulation selected. In one embodiment,the formulation is for general systemic administration, but in otherembodiments, the formulation comprises a targeting agent for targetedadministration to specific locations, receptors, cells, tissues, organs,or organ systems within a subject.

Provided is a composition comprising one or more of the herein providedoptimized peptides in a pharmaceutically acceptable carrier. Thus,provided is a composition comprising one or more any of the hereinprovided optimized peptides in a pharmaceutically acceptable carrier.Further, provided are compositions comprising one or more optimizedpeptides with an immune tolerance agent.

For example, provided is a composition comprising the peptide of SEQ IDNO:1 and in a pharmaceutically acceptable carrier. Another example is acomposition comprising the peptide of SEQ ID NO: 1 and Cyclosporine A ina pharmaceutically acceptable carrier. Another example is a compositioncomprising the peptide of SEQ ID NO: 2 in a pharmaceutically acceptablecarrier. Another example is a composition comprising the peptide of SEQID NO: 2 and Cyclosporine in a pharmaceutically acceptable carrier.Another example is a composition comprising a peptide of SEQ ID NO: 3 ina pharmaceutically acceptable carrier. Another example is a compositioncomprising the peptide of SEQ ID NO: 3 and Cyclosporine in apharmaceutically acceptable carrier. Another example is a compositioncomprising a peptide of SEQ ID NO: 4 in a pharmaceutically acceptablecarrier. Another example is a composition comprising the peptide of SEQID NO:4 and Cyclosporine in a pharmaceutically acceptable carrier.Another example is a composition comprising a peptide of SEQ ID NO: 5 ina pharmaceutically acceptable carrier. Another example is a compositioncomprising the peptide of SEQ ID NO: 5 and Cyclosporine in apharmaceutically acceptable carrier. Another example is a compositioncomprising a peptide of SEQ ID NO:6 in a pharmaceutically acceptablecarrier. Another example is a composition comprising the peptide of SEQID NO:6 and Cyclosporine in a pharmaceutically acceptable carrier.Another example is a composition comprising a peptide of SEQ ID NO:7 ina pharmaceutically acceptable carrier. Another example is a compositioncomprising the peptide of SEQ ID NO:7 and Cyclosporine in apharmaceutically acceptable carrier.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to a subject, along with the optimized peptide, withoutcausing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained. The carrier wouldnaturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art.

The compositions may be administered topically, orally, or parenterally.For example, the compositions can be administered extracorporeally,intracranially, intravaginally, intraanally, subcutaneously,intradermally, intracardiac, intragastric, intravenously,intramuscularly, by intraperitoneal injection, transdermally,intranasally, or by inhalation. As used herein, “intracranialadministration” means the direct delivery of substances to the brainincluding, for example, intrathecal, intracisternal, intraventricular ortrans-sphenoidal delivery via catheter or needle.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

As used herein, “topical intranasal administration” means delivery ofthe compositions into the nose and nasal passages through one or both ofthe nares and can comprise delivery by a spraying mechanism or dropletmechanism, or through aerosolization of the optimized peptide(s).Administration of the compositions by inhalant can be through the noseor mouth via delivery by a spraying or droplet mechanism. Delivery canalso be directly to any area of the respiratory system (e.g., lungs) viaintubation.

The exact amount of the compositions required will vary from subject tosubject, depending on the species, age, weight and general condition ofthe subject, the severity of the condition being treated, the particularoptimized peptide used, its mode of administration and the like. Thus,it is not possible to specify an exact amount for every composition.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutioncan be from about 5 to about 8, from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of compositionbeing administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,gels (e.g., poloxamer gel), drops, controlled-release compositions,timed release compositions, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. The disclosedcompositions can be administered, for example, in a microfiber, polymer(e.g., collagen), glasses, nanosphere, aerosol, lotion, cream, fabric,plastic, tissue engineered scaffold, matrix material, tablet, implantedcontainer, powder, oil, resin, wound dressing, bead, microbead,slow-release compounds, timed-release compounds, capsule, injectables,intravenous drips, pump device, silicone implants, or any bio-engineeredmaterials.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable. Pharmaceutically acceptablecarriers include fillers such as saccharides, for example lactose orsucrose, mannitol or sorbitol, cellulose preparations and/or calciumphosphates, for example tricalcium phosphate or calcium hydrogenphosphate, as well as binders such as starch paste, using, for example,maize starch, wheat starch, rice starch, potato starch, gelatin,tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariesare flow-regulating agents and lubricants, for example, silica, talc,stearic acid or salts thereof, such as magnesium stearate or calciumstearate, and/or polyethylene glycol. In one embodiment, dragee coresare provided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated saccharide solutions maybe used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquersolutions and suitable organic solvents or solvent mixtures. In order toproduce coatings resistant to gastric juices, solutions of suitablecellulose preparations such as acetylcellulose phthalate orhydroxypropylmethyl-cellulose phthalate, are used. Slow dissolvingpolymers such as poly(bis(p-carboxyphenoxy)-propane:sebacic acid—CCP:SA)may also be used to generate wafers or beads that control or time therelease of the composition. Dye stuffs or pigments may be added to thetablets or dragee coatings, for example, for identification or in orderto characterize combinations of active compound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules ornanoparticles which may optionally be mixed with fillers such aslactose, binders such as starches, and/or lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In one embodiment, thepeptides of this disclosure are dissolved or suspended in suitableliquids, such as fatty oils, or liquid paraffin, optionally withstabilizers.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Fatty oils may comprise mono-, di- or triglycerides. Mono-, di- andtriglycerides include those that are derived from C6, C8, C10, C12, C14,C16, C18, C20 and C22 acids. Exemplary diglycerides include, inparticular, diolein, dipalmitolein, and mixed caprylin-caprindiglycerides. Preferred triglycerides include vegetable oils, fish oils,animal fats, hydrogenated vegetable oils, partially hydrogenatedvegetable oils, synthetic triglycerides, modified triglycerides,fractionated triglycerides, medium and long-chain triglycerides,structured triglycerides, and mixtures thereof. Exemplary triglyceridesinclude: almond oil; babassu oil; borage oil; blackcurrant seed oil;canola oil; castor oil; coconut oil; corn oil; cottonseed oil; eveningprimrose oil; grapeseed oil; groundnut oil; mustard seed oil; olive oil;palm oil; palm kernel oil; peanut oil; rapeseed oil; safflower oil;sesame oil; shark liver oil; soybean oil; sunflower oil; hydrogenatedcastor oil; hydrogenated coconut oil; hydrogenated palm oil;hydrogenated soybean oil; hydrogenated vegetable oil; hydrogenatedcottonseed and castor oil; partially hydrogenated soybean oil; partiallysoy and cottonseed oil; glyceryl tricaproate; glyceryl tricaprylate;glyceryl tricaprate; glyceryl triundecanoate; glyceryl trilaurate;glyceryl trioleate; glyceryl trilinoleate; glyceryl trilinolenate;glyceryl tricaprylate/caprate; glyceryl tricaprylate/caprate/laurate;glyceryl tricaprylate/caprate/linoleate; and glyceryltricaprylate/caprate/stearate.

In one embodiment, the triglyceride is the medium chain triglycerideavailable under the trade name LABRAFAC CC. Other triglycerides includeneutral oils, e.g., neutral plant oils, in particular fractionatedcoconut oils such as known and commercially available under the tradename MIGLYOL, including the products: MIGLYOL 810; MIGLYOL 812; MIGLYOL818; and CAPTEX 355. Other triglycerides are caprylic-capric acidtriglycerides such as known and commercially available under the tradename MYRITOL, including the product MYRITOL 813. Further triglyceridesof this class are CAPMUL MCT, CAPTEX 200, CAPTEX 300, CAPTEX 800, NEOBEEM5 and MAZOL 1400.

Pharmaceutical compositions comprising triglycerides may furthercomprise lipophilic and/or hydrophilic surfactants which may form clearsolutions upon dissolution with an aqueous solvent. One such surfactantis tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS).Examples of such compositions are described in U.S. Pat. No. 6,267,985.

Suitable formulations for parenteral administration include aqueoussolutions of hamster Reg3 gamma and optimized hamster Reg3 gammapeptides and optionally immune tolerance agents in water-soluble form,for example, water-soluble salts and alkaline solutions. In addition,suspensions of hamster Reg3 gamma and optimized hamster Reg3 gammapeptides and optionally immune tolerance agent(s) as appropriate oilyinjection suspensions may be administered. Suitable lipophilic solventsor vehicles include fatty oils, for example, sesame oil, or syntheticfatty acid esters, for example, ethyl oleate or triglycerides orpolyethylene glycol-400. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension include, forexample, sodium carboxymethyl cellulose, sorbitol, and/or dextran.Optionally, the suspension may also contain stabilizers.

The topical compositions may be formulated as oils, creams, lotions,ointments and the like by choice of appropriate carriers. Suitablecarriers include vegetable or mineral oils, white petrolatum (white softparaffin), branched chain fats or oils, animal fats and high molecularweight alcohol (greater than C12). Emulsifiers, stabilizers, humectantsand antioxidants may also be included as well as agents imparting coloror fragrance, if desired. Additionally, transdermal penetrationenhancers can be employed in these topical formulations. Examples ofsuch enhancers can be found in U.S. Pat. Nos. 3,989,816 and 4,444,762.

Creams may be formulated from a mixture of mineral oil, self-emulsifyingbeeswax and water in which hamster Reg3 gamma and optimized hamster Reg3gamma peptides and optionally immune tolerance agents, dissolved in asmall amount of an oil such as almond oil, is admixed. A typical exampleof such a cream is one which includes about 40 parts water, about 20parts beeswax, about 40 parts mineral oil and about 1 part almond oil.

Ointments may be formulated by mixing a suspension of hamster Reg3 gammaimmune or optimized hamster Reg3 gamma peptides in a vegetable oil suchas almond oil with warm soft paraffin and allowing the mixture to cool.A typical example of such an ointment is one which includes about 30%almond oil and about 70% white soft paraffin by weight.

Lotions may be conveniently prepared by preparing a suspension of thehamster Reg3 gamma, and optimized hamster Reg3 gamma peptides andoptionally immune tolerance agent(s) in a suitable high molecular weightalcohol such as propylene glycol or polyethylene glycol.

Suitable routes of administering the pharmaceutical preparations includeoral, rectal, topical (including dermal, buccal and sublingual),vaginal, parenteral (including subcutaneous, intramuscular, intravenous,intradermal, intrathecal, intratumoral, and epidural) and bynaso-gastric tube. It will be understood by those skilled in the artthat the preferred route of administration will depend upon thecondition being treated and may vary with factors such as the conditionof the recipient.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories, which consist of a combination of one ormore of the peptides of this disclosure with a suppository base.Suitable suppository bases are, for example, natural or synthetictriglycerides, or paraffin hydrocarbons. In addition, it is alsopossible to use gelatin rectal capsules which consist of the peptides ofthis disclosure with a base. Possible base materials include, forexample, liquid triglycerides, polyethylene glycols, or paraffinhydrocarbons.

Effective dosages and schedules for administering the compositions maybe determined empirically, and making such determinations is within theskill in the art. The dosage ranges for the administration of thecompositions are those large enough to produce the desired effect inwhich the symptoms of the disorder are affected. The dosage should notbe so large as to cause adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage will vary with the age, condition, sex and extent of the diseasein the patient, route of administration, or whether other drugs areincluded in the regimen, and can be determined by one of skill in theart. The dosage can be adjusted by the individual doctor in the event ofany counter indications. Dosage can vary, and can be administered in oneor more dose administrations daily, for one or several days. Guidancecan be found in the literature for appropriate dosages for given classesof pharmaceutical products. The range of dosage largely depends on theapplication of the compositions herein, severity of condition, and itsroute of administration.

For example, in applications as a laboratory tool for research, theoptimized peptide compositions can be used in doses as low as 0.01% w/v.Significantly higher concentrations of the compositions by themselves orin combination with other compounds may be used in applications likecancer/tumor therapy. Thus, upper limits of the provided polypeptidesmay be up to 2-5% w/v or v/v if given as an initial bolus delivered.Recommended upper limits of dosage for parenteral routes ofadministration for example intramuscular, intracerebral,intracardicardiac and intraspinal could be up to 1% w/v or v/v dependingon the severity of the disease. This upper dosage limit may vary byformulation, depending for example on how the polypeptide(s) is combinedwith other agents promoting its action or acting in concert with thepolypeptide(s).

For continuous delivery of the provided polypeptides, for example, incombination with an intravenous drip, upper limits of 0.01 g/Kg bodyweight over time courses determined by the doctor based on improvementin the condition can be used. In another example, upper limits ofconcentration of the provided nucleic acids delivered internally forexample, intramuscular, intracerebral, intracardicardiac and intraspinalwould be 50-100 μg/ml of solution. Again, the frequency would bedetermined by the Doctor based on improvement.

The optimized peptides of this disclosure may be optionally formulatedwith an immune tolerance agent. The immune tolerance agent may bepresent at a concentration of 0.01% to 10% w/v, depending on whichimmune tolerance agent is used and the type of vehicle. In oneembodiment, the immune tolerance agent is Cyclosporine. For example,Cyclosporine may be present in a concentration of 1 mg/ml to 200 mg/ml.The optimized peptides of this disclosure may be formulated with animmune tolerance agent using any vehicle described herein.

In other embodiments of the present invention, provided are methods oftreating a pathology associated with impaired pancreatic function in asubject in need of such treatment. The method may comprise the step ofadministering one or more agents for stimulating pancreatic islet cellregeneration in addition to the optimized hamster Reg3gamma peptide. Inone aspect of this embodiment, the agents are selected from hamster Reg3gamma or hamster Reg3 gamma-related peptides other than Optimized Reg3gamma, amylin/Pramlintide (SYMLIN™), exendin-4 (EXENATIDE™), GIP, GLP-1,GLP-1 receptor agonists, GLP-1 analogs, hamster INGAP peptide andrelated peptides, Liraglutide (NN2211), and a dipeptidyl peptidaseinhibitor, which blocks the degradation of GLP-1.

In another embodiment, methods of treating a pathology associated withimpaired pancreatic function in a subject in need of such treatment areprovided. The method may comprises one or more of the steps of (1)intensifying glycemic control; (2) administering oral vitamin D3(cholecalciferol) to maintain 25-hydroxyvitamin levels above 40 ng/ml;(3) administering one or more immune therapies for protecting new isletcell formation, including administration of immunosuppressive agents;(4) administering selenium 5) administering Optimized hamster Reg3gammapeptide in combination with insulin but decreasing the insulinadministered over time; and (5) repeatedly administering a therapy forprotection of islets, preferably on a 3 to 24 month basis, depending onthe selected immune therapy, in addition to the step of administeringoptimized hamster Reg3gamma peptide.

In another embodiment, methods of treating a pathology associated withimpaired pancreatic function in a subject in need of such treatment areprovided. The method may comprise one or more of the steps of: (1)intensifying glycemic control; (2) administering oral vitamin D3(cholecalciferol) to maintain 25-hydroxyvitamin levels above 40 ng/ml;(3) administering an agent for stimulating pancreatic islet regenerationin addition to Optimized hamster Reg gamma peptide, including but notlimited to the 15 amino-acid hamster Reg3 gamma peptide and 15 aminoacid hamster Reg gamma analogs other than Optimized hamster Reg3 gammapeptide; (4) co-administering an agent selected from the groupconsisting of amylin/Pramlintide (SYMLIN™), exendin-4 (EXENATIDE™;BYETTA™), Gastrin, Epidermal Growth Factor and Epidermal Growth Factoranalog GIP, GLP-1, GLP-1 receptor agonists, GLP-1 analogs, INGAP,Liraglutide (NN2211), and a dipeptidyl peptidase IV inhibitor, whichblocks the degradation of GLP-1; and (5) reducing, or tapering off,administration of another diabetes therapy.

In another embodiment, methods of treating a pathology associated withimpaired pancreatic function in a subject in need of such treatment areprovided. The method may comprise in addition to administering theoptimized 15 amino acid hamster Reg3gamma peptide, the step ofadministering one or more agents that inhibit, block, or destroy theautoimmune cells that target pancreatic islets. Such therapies aretermed “immune therapies” above. In various aspects of this embodiment,the agents that inhibit, block, or destroy the autoimmune cells thattarget pancreatic islets are selected from the group consisting ofCyclosporine A, Anti CD-3 antibodies including hOKT3γ1(Ala-Ala) andChAglyCD3 that target the immune response and specifically block theT-lymphocytes that cause beta cell death in type 1 diabetes; Sirolimus(Rapamycin); Tacrolimus (FK506); a heat-shock protein 60 (Diapep277); ananti-Glutamic Acid Decarboxylase 65 (GAD65) vaccine; MycophenolateMofetil alone or in combination with Daclizumab; the anti-CD20 agent,Rituximab; Campath-1H (Anti-CD52 Antibody), lysofylline; antithymocyteglobulin (ATG), Vitamin D; IBC-VSO vaccine, which is a synthetic,metabolically inactive form of insulin designed to prevent pancreaticbeta-cell destruction; interferon-alpha; and a vaccine using CD4⁺CD25⁺antigen-specific regulatory T cells. These or similar agents can be usedin the combination therapies provided by the invention that utilizeregulatory T cells either directly or through the use of immunotherapyto arrest the destruction of insulin-producing cells.

In another embodiment of the present invention, methods of treating apathology associated with impaired pancreatic function in a subject inneed of such treatment, wherein at least one symptom of the pathologyassociated with impaired pancreatic function is treated or reduced as aresult of the administration of the optimized 15 amino-acid hamster Reg3gamma peptide are provided. In one aspect of this embodiment, thesymptom is selected from low levels of insulin or insulin activity,insulin resistance, hyperglycemia, hemoglobin A1C level greater than6.0%, frequent urination, excessive thirst, extreme hunger, unusualweight loss or gain, being overweight, increased fatigue, irritability,blurry vision, genital itching, odd aches and pains, dry mouth, dry oritchy skin, impotence, vaginal yeast infections, poor healing of cutsand scrapes, excessive or unusual infections, loss or worsening ofglycemic control, fluctuations in blood glucose, fluctuations in bloodglucagon, and fluctuations in blood triglycerides, with hyperglycemiaultimately leading to microvascular and macrovascular complications,which include visual symptoms that lead to blindness, accelerated kidneyimpairment that can lead to renal failure necessitating dialysis andkidney transplant and neuropathy leading to foot ulcers and amputations.Additionally, recent studies have demonstrated both microvascular andmacrovascular/cardiovascular risk reduction among type 1 diabetespatients who have improved glycemic control.

In another embodiment, methods of treating a pathology associated withimpaired pancreatic function in a subject in need of such treatment areprovided. The pathology associated with impaired pancreatic function isany one of type 1 diabetes, new onset type 1 diabetes, type 2 diabetes,latent autoimmune diabetes of adulthood, pre-diabetes, impaired fastingglucose, impaired glucose tolerance, insulin resistant syndrome,metabolic syndrome/dysmetabolic syndrome, being overweight, obesity,hyperlipidemia, hypertriglyceridemia, eating disorders, anovulatorycycles and polycystic ovarian syndrome.

Embodiments of the invention also provide antibodies which selectivelybind to the optimized 15 amino acid Reg3 gamma peptide. In oneembodiment, the antibody is a monoclonal antibody. In anotherembodiment, the antibody is a polyclonal antibody. Such antibodies canbe used in diagnostic methods provided by the invention, which methodscomprise detecting the optimized 15 amino acid hamster Reg3gamma peptidelevels in the serum or tissue of a mammal. In one embodiment thediagnostic method is used to monitor treatment with the optimized 15amino acid Reg3 hamster gamma peptide to ensure that therapeuticallyeffective levels are being achieved in a patient receiving such therapy.

Embodiments of the invention also provide kits for treating a patienthaving type 1 or type 2 diabetes or other condition in which there areaberrant insulin levels, perturbation in glucose metabolism or insulinresistance, comprising a therapeutically effective dose of the 15 aminoacid optimized hamster Reg 3 gamma peptide and optionally at least oneagent for stimulating GLP-1 receptors or enhancing GLP-1 levels,promoting beta cell regeneration, increased satiety, decreased foodintake and weight loss, either in the same or separate packaging, andinstructions for its use. Further embodiments provide a kit formeasuring optimized hamster Reg3 gamma levels in a sample, the kitcomprising an optimized hamster Reg3 gamma-specific antibody andoptionally optimized hamster Reg3 gamma and optionally a labeling means.

Embodiments of the present invention provide detailed strategies foroptimizing the stability and solubility of a 15 amino-acid hamster Reg3gamma for improved use as a therapeutic agent and are a peptide fragmentof the hamster protein regenerating islet-derived 3 gamma protein.Embodiments of the invention also provide pharmaceutical compositionsand therapies for the treatment of pancreatic dysfunction, includingtype 1 and type 2 diabetes, with such compositions. In one embodiment,these compositions comprise an optimized 15 amino acid hamster Reg 3gamma. In another embodiment, these compositions include optimizedhamster Reg3 gamma and other agents that affect glucose metabolism.Included among these other agents are agents that are involved inpancreatic islet neogenesis and agents that inhibit, block, or destroythe autoimmune cells that target pancreatic islet cells. In oneembodiment, the therapies of the invention are practiced byadministering a therapeutically effective dose of the hamster optimizedReg3 gamma peptide to a mammal in need of such therapy. In anotherembodiment, the therapies of the invention are practiced byadministering a therapeutically effective dose of the hamster optimizedReg 3 gamma peptide to a mammal in need of such therapy in combinationwith another agent (such as a hormone or compound) that affects glucosemetabolism, including but not limited to hormones or compounds that areinvolved in beta cell regeneration, satiety, and gastric emptying, suchas GLP-1, GIP, GLP-1 receptor analogs, GLP-1 analogs, and DipeptidylPeptidase-4 Inhibitors, which prevent destruction of GLP-1, and agentsthat inhibit, block, or destroy the autoimmune cells that targetpancreatic cells. In this latter embodiment, the Optimized hamster Reg3gamma and the other agent may be administered separately or may first beadmixed to provide a combination composition of the invention andadministered simultaneously.

An exemplary composition comprises 100 mg optimized Blocked Reg3 gammapeptide (SEQ ID NO:2) in a vial for intravenous injection. The vialcontains inactive ingredients including mannitol (600 mg), L-threonine(160 mg), polysorbate 80 (120 mg), sodium citrate dihydrate (235 mg),and hydrochloric acid (160 mg). When reconstituted with 41 mL SterileWater for Injection, the resulting solution will contain 2.5 mg/mloptimized Reg3 gamma peptide (SEQ ID NO:2).

Another exemplary composition comprises 250 mg optimized Cys N-terminalReg3 gamma peptide (SEQ ID NO:3) in 100 mL of Lactated Ringers toprovide a solution of 2.5 mg/ml optimized Reg3 gamma peptide (SEQ IDNO:3).

Another exemplary composition comprises 200 mg optimized Cys Dimer Reg3gamma peptide (SEQ ID NO:4) in 100 mL of 5% dextrose in 0.9% sodiumchloride to provide a solution of 2.0 mg/ml optimized Reg3 gamma peptide(SEQ ID NO:4).

Another exemplary composition comprises 500 mg optimized Cys BlockedDimer Reg3 gamma peptide (SEQ ID NO:5) in 1000 ml in 0.9% sodiumchloride to provide a solution of 0.5 mg/ml optimized Reg3 gamma peptide(SEQ ID NO:5).

Another exemplary composition comprises 100 mg optimized Cys PEG Reg3gamma peptide (SEQ ID NO:6) in 1 ml MIGLYOL 810 enclosed in a gelatincapsule.

Another exemplary composition comprises 100 mg optimized Cys Blocked PEGReg3 gamma peptide (SEQ ID NO:7) in 1 ml LABRAFAC CC enclosed in agelatin capsule.

In another embodiment, the invention provides a combination productcomprising at least one optimized Reg3 gamma peptide combined with animmune tolerance agent. The combination product may be used in type 1and 2 diabetes, PreDiabetes or diseases of insulin deficiency, beta celldeficiency, insulin resistance and impaired glucose metabolism. Oneexemplary composition comprises 100 mg Reg3 gamma peptide (SEQ ID NO:1)and 5 g Cyclosporine in 50 ml distilled water to provide a solution of 2mg/ml Reg3 gamma peptide (SEQ ID NO:1) and 100 mg/ml Cyclosporine.

Another exemplary composition comprises 100 mg Reg3 gamma peptide (SEQID NO:1) and 5 g Cyclosporine in 50 ml 0.9% NaCl; to provide a solutionof 2 mg/ml Reg3 gamma peptide (SEQ ID NO:1) and 100 mg/ml Cyclosporine.

Another exemplary composition comprises 150 mg optimized Blocked Reg3gamma peptide (SEQ ID NO:2) and 5 g Cyclosporine in 50 ml 0.9% NaCl toprovide a solution of 3 mg/ml optimized Reg 3 gamma peptide (SEQ IDNO:2) and 100 mg/ml Cyclosporine.

Another exemplary composition comprises 200 mg optimized Cys Reg3 gammapeptide (SEQ ID NO:3) and 5 g Cyclosporine in 50 ml 0.9% NaCl to providea solution of 4 mg/ml optimized Reg 3 gamma peptide (SEQ ID NO:3) and100 mg/ml Cyclosporine.

Another exemplary pharmaceutical composition comprises 250 mg CysDimerReg3 gamma peptide (SEQ ID NO:4) and 5 g Cyclosporine in 50 ml 0.9% NaClto provide a solution of 5 mg/ml optimized Reg 3 gamma peptide (SEQ IDNO:4) and 100 mg/ml Cyclosporine.

Another exemplary pharmaceutical composition comprises 150 mg CysBlockedDimer Reg3 gamma peptide (SEQ ID NO:5) and 5 g Cyclosporine in 50 mlLactated Ringer's to provide a solution of 3 mg/ml optimized Reg 3 gammapeptide (SEQ ID NO:5) and 100 mg/ml Cyclosporine.

Another exemplary pharmaceutical composition comprises 200 mg CysPEGReg3 gamma peptide (SEQ ID NO:6) and 5 g Cyclosporine in 50 ml 0.9%Lactated Ringer's to provide a solution of 4 mg/ml optimized Reg 3 gammapeptide (SEQ ID NO:6) and 100 mg/ml Cyclosporine.

Another exemplary pharmaceutical composition comprises 250 mgCysBlockedPEG Reg3 gamma peptide (SEQ ID NO:7) and 5 g Cyclosporine in50 ml Lactated Ringer's to provide a solution of 5 mg/ml optimized Reg 3gamma peptide (SEQ ID NO:7) and 100 mg/ml Cyclosporine.

Another exemplary pharmaceutical composition of the present inventioncomprises 60 mg Reg3 gamma peptide (SEQ ID NO:1) and 100 mg Cyclosporineenclosed in a hard gelatin capsule with lactose as a filler.

Another exemplary pharmaceutical composition of the present inventioncomprises 50 mg Blocked Reg3 gamma peptide (SEQ ID NO:2), 100 mgCyclosporine, QS to 1 ml the pharmaceutically acceptable carrierLABRASOL (Gattefosse S A), which is PEG-8 caprylic/capric glycerides,enclosed in a soft gelatin capsule.

Another exemplary pharmaceutical composition of the present inventioncomprises 100 mg N-terminal Cys Reg3 gamma peptide (SEQ ID NO:3), 50 mgCyclosporine, QS to 1 ml the pharmaceutically acceptable carrier Miglyol812N (medium chain triglycerides), enclosed in a hard gelatin capsule.

Another exemplary pharmaceutical composition of the present inventioncomprises 75 mg CysDimer Reg3 gamma peptide (SEQ ID NO:4), 100 mgCyclosporine, and alcohol, USP, absolute, 12.7% v/v, enclosed in a softgelatin capsule.

Another exemplary pharmaceutical composition is an oral solution whereineach ml contains 50 mg CysBlockedDimer Reg3 gamma peptide (SEQ ID NO:5),100 mg Cyclosporine, alcohol, Ph. Helv. 12.5% by volume dissolved in anolive oil, Ph. Helv./Labrafil M 1944 CS (polyoxyethylated oleicglycerides) vehicle which must be further diluted with milk, chocolatemilk, or orange juice before oral administration.

Another exemplary pharmaceutical composition is an oral suspensionwherein each ml contains 3 mg CysPEG Reg3 gamma peptide (SEQ ID NO:6)and 5 mg Cyclosporine, QS to 1 ml 8.4% sodium bicarbonate (aqueoussolution).

Another exemplary composition is 30 mg CysBlockedPEG Reg3 gamma peptide(SEQ ID NO:7) and 50 mg Cyclosporine formulated in a pill comprisingpharmaceutically acceptable carriers such as fillers (e.g. saccharides,cellulose preparations and/or calcium phosphates).

Another exemplary pharmaceutical composition of the present inventioncomprises 50 mg human Reg3 gamma peptide (SEQ ID NO:1), 100 mgCyclosporine, and alcohol, USP, absolute, 12.8% v/v, enclosed in a softgelatin capsule.

Example 1 The Optimized Hamster Reg3 Gamma Peptide Used withCyclosporine for Insulin Independence Among Type 1 Diabetes Patients

The combination of an immune tolerance agent (e.g. Cyclosporineinitially dosed at 7.5 mg/kg/day in divided dosages at breakfast anddinner and based on peak and trough levels, the dosage will be modifiedto optimize immune tolerance and limit side effects) with optimizedhamster Reg3gamma (each of SEQ ID NOS:2-7) dosed at 30 mg per day givenin two divided given subcutaneously (15 mg per dosage) for children lessthan 11 years old weighing 66 pounds or less. For children older than 11years and weighing more than 66 pounds optimized hamster Reg3gamma (eachof SEQ ID NOS:2-7) will be dosed as 60 mg per day given subcutaneouslyin two divided dosage of 30 mg each. Exogenous insulin dosages, whetherby injection or pump, are decreased and able to be tapered off basedupon glucose levels before meals and fasting. Modifications made inlowering insulin, will be made based on whether the patient demonstrateshigh or low fasting glucose levels, commonly impacted by a basal insulinvs. the patient having high or low pre-meal glucose levels, which maylikely reflect the dosing of insulin at the prior meal, whereas, the2-hour postprandial glucose levels reflects the insulin given prior tothe meal.

Example 2 Optimized Hamster Reg3 Gamma Peptide Used for InsulinIndependence Among Type 2 Diabetes

Thirty milligrams of Optimized Reg3 gamma peptide (each of SEQ IDNOS:2-7) will be given subcutaneously per day will be given in twodivided (15 mg per dosage) for children less than 11 years old weighing66 pounds or less, and for children older than 11 years and weighingmore than 66 pounds. Optimized hamster Reg3 gamma peptide (each of SEQID NOS:2-7) will be dosed as 60 mg per day subcutaneously given in twodivided dosage of 30 mg each to results in insulin independence. Foradults, optimized hamster Reg3 gamma peptide will be given as 60 mgtwice daily subcutaneously resulting in insulin independence. Exogenousinsulin dosages, whether by injection or pump are decreased based onglucose levels before meals and fasting. Exogenous insulin dosages,whether by injection or pump, are decreased and able to be tapered offbased upon glucose levels before meals and fasting. Modifications madein lowering insulin, will be made based on whether the patientdemonstrates high or low fasting glucose levels, commonly impacted by abasal insulin vs. the patient having high or low pre-meal glucoselevels, which may likely reflect the dosing of insulin at the priormeal, whereas, the 2-hour postprandial glucose levels reflects theinsulin given prior to the meal.

Based upon glucose levels, other diabetes agents such as sulfonylureas,metformin, meglitinides, GLP-1 receptor analogs, DPP-4 inhibitors,thiazolidinediones, SGLT2 inhibitors, anti-inflammatory agents andpramlintide may also be tapered based on glucose levels and hemoglobinA1C. Metformin, thiazolidinediones, SGLT2 inhibitors work as basalglucose lowering agents, whereas, sulfonylureas, GLP-1 receptor analogs,DPP-4 inhibitors, meglitinides work to reduce postprandial glucoselevels, thus modifications made in lowering these agents will be basedon whether the patient demonstrates high or low fasting glucose levels,commonly impacted by a basal agent vs. the patient has high or lowpre-meal glucose levels, which may likely reflect under dosing of adiabetes medication prior to the previous meal.

Example 3 Optimized Hamster Reg3 Gamma Peptide Used for ReducingDiabetes Medications Requirements Among Type 2 Diabetes

Optimized hamster Reg3 gamma peptide (each of SEQ ID NOS:2-7) will bedosed at 30 mg per day given in two divided (15 mg per dosage) forchildren less than 11 years old weighing 66 pounds or less, and forchildren older than 11 years and weighing more than 66 pounds optimizedhamster Reg3 gamma peptide (each of SEQ ID NOS:2-7) will be dosed as 60mg per day given in two divided dosage of 30 mg subcutaneously toresults in insulin independence. For adults, optimized hamster Reg3gamma peptide (each of SEQ ID NOS:2-7) will be given as 60 mg twicedaily by subcutaneous injection. Optimized hamster Reg3 gamma peptide(each of SEQ ID NOS:2-7) given in divided dosages in one capsule/pill orin on oral suspension may result in the need to diminish dosages ofother diabetes medications utilized and such medications may potentiallybe tapered off. Medications include: sulfonylureas, metformin,meglitinides, GLP-1 receptor analogs, DPP-4 inhibitors,thiazolidinediones, SGLT2 inhibitors, anti-inflammatory agents andpramlintide may also be tapered based on glucose levels and hemoglobinA1C. Metformin, thiazolidinediones, SGLT2 inhibitors work as basalglucose lowering agents, whereas, sulfonylureas, GLP-1 receptor analogs,DPP-4 inhibitors, meglitinides work to reduce postprandial glucoselevels, thus modifications made in lowering these agents will be basedon whether the patient demonstrates high or low fasting glucose levels,commonly impacted by a basal agent vs. the patient has high or lowpre-meal glucose levels, which may likely reflect under dosing of adiabetes medication prior to the previous meal. Modifications made tothe diabetes medication regimen will be made based on whether thepatient glucose levels and the need to adjust the basal or postprandialagent will be made.

Example 4 Optimized Hamster Reg3 Gamma Peptide Used for Drug Naive Type2 Diabetes

Optimized hamster Reg3 gamma peptide (each of SEQ ID NOS:2-7) dosed at30 mg per day given in two divided (15 mg per dosage) for children lessthan 11 years old weighing 66 pounds or less. For children older than 11years and weighing more than 66 pounds, optimized hamster Reg3 gammapeptide (each of SEQ ID NOS:2-7) will be dosed as 60 mg per day given intwo divided dosage of 30 mg each and may be delivered subcutaneously toresults in insulin independence. For adults, optimized hamster Reg3gamma peptide will be given as 60 mg twice daily by mouth in pill ororal suspension. Optimized hamster Reg3 gamma peptide (each of SEQ IDNOS:2-7) given in divided dosages in one capsule/pill or in on oralsuspension results in normalization of blood glucose as measured byfasting glucose and hemoglobin A1C levels. Among a newly diagnosed orpreviously diagnosed type 2 diabetes patient who is currently on nopharmaceutical treatment for diabetes, optimized hamster Reg3 gammapeptide (each of SEQ ID NOS:2-7) is utilized among patients with type 2diabetes with a primary endpoint of glucose levels and Hemoglobin A1C inthe normal range with the glucose goals would be 100 mg/dL range beforemeals and 140 mg/dL two hours after meals.

Example 5 Optimized Hamster Reg3 Gamma Peptide Used for Ex VivoGeneration of Beta Cells and Provided to Patients with Labile Type 1with Cyclosporine for Insulin Independence and Type 2 Diabetes forInsulin Independence without an Immune Tolerance Agent Required

Optimized hamster Reg3 gamma peptide (each of SEQ ID NOS:2-7) and/oragents that bind to the human Reg Receptor are used for the ex vivotransformation of new beta cells from pluripotent stem cells includingembryonic cells, adult somatic stem cells, human adult bone-marrowderived stem cells, umbilical cord stems cells, mesenchymal stem cells,human amniotic membrane-derived mesenchymal cells, mammalian stem cells,mammalian stem cells, ectodermal stem cells or other stem cells and mayinclude resident populations of endogenous stem cells that exist withinthe adult pancreas. The new beta cells are then administered to patientswith new and existing type 1 and 2 diabetes, PreDiabetes or diseases ofinsulin deficiency, beta cell deficiency, insulin resistance andimpaired glucose metabolism, with routes of delivery to include, but arenot limited to the portal and umbilical vein, oral, intravenous,subcutaneous delivery with and without organ specific targeting and mayinclude direct administration to the pancreas or liver. Patientsreceiving ex vivo formulated beta cells will require an immune toleranceagent to prevent autoimmune attack of the newly received beta cells. Forexample, patients will receive 7.5 mg/kg/day of Cyclosporine in divideddosages prior to receiving the ex vivo generated beta cells with dosagesof Cyclosporine adjusted based upon peak and trough levels to optimizeefficacy and reduce risks of side effects.

What is claimed is:
 1. A pharmaceutical composition comprising a peptidehomodimer comprising SEQ ID NO:
 4. 2. The pharmaceutical composition ofclaim 1, further comprising: an immune tolerance agent selected from thegroup consisting of: a. Cyclosporine A, b. hOKT3y1(Ala-Ala), c.ChAglyCD3, d. Sirolimus (Rapamycin), e. Tacrolimus (FK506), f. aheat-shock protein 60 (Diapep277), g. anti-Glutamic Acid Decarboxylase65 (GAD65) vaccine, h. Mycophenolate Mofetil alone or in combinationwith Daclizumab, i. anti-CD20 agent (Rituximab), j. Campath-1H(Anti-CD52 Antibody), lysofylline k. antithymocyte globulin (ATG), l.Vitamin D, m. IBC-VSO vaccine, n. interferon-alpha, and o. vaccine usingCD4+CD25+ antigen-specific regulatory T cells, and p. Lysofylline. 3.The pharmaceutical composition of claim 1, comprising the peptidehomodimer set forth in SEQ ID NO: 4 and any combination of acetylationat an N-terminus, amidation at a C-terminus, and/or PEGylation at eitherterminus.
 4. The pharmaceutical composition of claim 1, comprising thepeptide homodimer set forth in SEQ ID NO: 4 is acetylated at anN-terminus and amidated at a C-terminus.
 5. The pharmaceuticalcomposition of claim 1, comprising the peptide homodimer set forth inSEQ ID NO: 4 is acetylated at the N-terminus and amidated at theC-terminus.
 6. The pharmaceutical composition of claim 1, wherein thecomposition is formulated for systemic administration.
 7. Thepharmaceutical composition of claim 1, wherein the composition comprisesa targeting agent for targeted administration to specific locations,receptors, cells, tissues, organs, or organ systems.
 8. Thepharmaceutical composition of claim 2, wherein the immune toleranceagent is Cyclosporine.
 9. The pharmaceutical composition of claim 2,wherein the composition is formulated for systemic administration. 10.The pharmaceutical composition of claim 2, wherein the compositioncomprises a targeting agent for targeted administration to specificlocations, receptors, cells, tissues, organs, or organ systems.
 11. Acomposition comprising a peptide homodimer comprising SEQ ID NO: 4 in apharmaceutically acceptable carrier.
 12. The composition of claim 11,further comprising: an immune tolerance agent selected from the groupconsisting of: a. Cyclosporine A, b. hOKT3y1(Ala-Ala), c. ChAglyCD3, d.Sirolimus (Rapamycin), e. Tacrolimus (FK506), f. a heat-shock protein 60(Diapep277), g. anti-Glutamic Acid Decarboxylase 65 (GAD65) vaccine, h.Mycophenolate Mofetil alone or in combination with Daclizumab, i.anti-CD20 agent (Rituximab), j. Campath-1H (Anti-CD52 Antibody),lysofylline k. antithymocyte globulin (ATG), l. Vitamin D, m. IBC-VSOvaccine, n. interferon-alpha, and o. vaccine using CD4+CD25+antigen-specific regulatory T cells, and p. Lysofylline.
 13. Thecomposition of claim 12, wherein the immune tolerance agent isCyclosporine.
 14. The composition of claim 11, comprising the peptidehomodimer set forth in SEQ ID NO: 4 and any combination of acetylationat an N-terminus, amidation at a C-terminus, and/or PEGylation at eitherterminus.
 15. The composition of claim 11, comprising the peptidehomodimer set forth in SEQ ID NO: 4 is acetylated at an N-terminus andamidated at a C-terminus.
 16. The composition of claim 11, comprisingthe peptide homodimer set forth in SEQ ID NO: 4 is acetylated at theN-terminus and amidated at the C-terminus.
 17. The composition of claim11, wherein the composition is formulated for systemic administration.18. The composition of claim 11, wherein the composition comprises atargeting agent for targeted administration to specific locations,receptors, cells, tissues, organs, or organ systems.
 19. The compositionof claim 12, wherein the immune tolerance agent is Cyclosporine.
 20. Thecomposition of claim 12, wherein the composition is formulated forsystemic administration.